Project Summary/ Abstract Studies conducted over the past decade have helped uncover key regulators of fetal hemoglobin (HbF) expression, including the major HbF repressor, BCL11A. Despite these considerable advances, which have largely been inspired by studies of common and rare human genetic variation, many questions remain. Specifically, the precise mechanisms by which HbF is regulated are unclear. Studies of HbF regulation have been constrained by two challenges. First, nearly all reported genetic mutations/ deletions with large effects upon HbF expression have generally been assessed in one or a limited number of individuals, restricting the inferences that can be made from such findings. Second, existing cellular models for studies of HbF regulation have numerous limitations and do not faithfully recapitulate observations made in vivo or in primary cells. To overcome these two issues, here we propose to utilize genetic data from a large population-based study we have already conducted and couple this with a new single cell-derived functional assay using cutting-edge genome editing tools to determine the impact of naturally-occurring deletions and predicted cis-regulatory elements on HbF levels. We have studied variation in HbF levels in a population of over 86,000 individuals and focused our genetic analysis on rare individuals harboring elevated HbF. This has enabled us to identify several mutations and deletions causing increased HbF expression. However, there is considerable variation noted even in individuals with the same deletion/ mutation, suggesting that a complex genetic architecture determines HbF levels. We plan to employ a single cell-derived genome editing functional assay to assess the impact of creating specific perturbations in an isogenic setting on HbF expression. We will couple this assay with innovative genetic mapping approaches using our rich population genetic dataset. These studies will enable us to nominate novel cis-regulatory elements that impact HbF levels, as well as the trans-acting factors that function through these cis-elements. Success in the proposed studies should enable new insights into HbF regulation and facilitate the development of curative approaches for sickle cell disease and b-thalassemia.